Effective antitumor immunity involves successful priming of tumor-specific T-cells by dendritic cells (DC) in the lymph node (LN), followed by trafficking, infiltration and sustained elimination of tumor cells by T-cells. Immunotherapies aim to facilitate or further invigorate these processes, but at which anatomic sites these therapies act and in which specific patients remains unknown. We and others have identified macrophages as key mediators of immune suppression in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are capable of negating the effectiveness of multiple conventional- and immune-targeted anticancer therapies. Several current therapeutic strategies aim to deplete or reprogram macrophages; however, as these drugs act systemically, their precise mechanism and site of action remains unclear. Besides TAMs, macrophages in the LN have been identified as potent immune modulatory cells in diverse settings. Using a novel method that allows for the specific interrogation of LN-macrophages, we aimed to investigate the role of LN macrophages in regulating DC-induced anti-tumor immunity. To determine the immune modulatory functions of the tumor on the LN, we extensively characterized the immune contexture and phenotype of the tumor-draining lymph node (TDLN) compared to a distant non-tumor draining lymph node (non-TDLN) using multicolor flow- and histo-cytometry, in an orthotopic mouse model of peritoneal mesothelioma. In addition, the effects of systemic macrophage depletion using an CSF1R-kinase inhibitor or clodronate encapsulated liposomes (CEL) were evaluated. We developed a method to specifically deplete TDLN-macrophages while leaving TAMs and other tissue macrophages intact, by intrapleural (i.pl) injection of low-dose CEL. Using this model, we investigated the immune-regulatory properties of LN-macrophages following adoptive transfer of activated, tumor-loaded DCs in the TDLN by both flow- and histo-cytometry. Comparison of the TDLN and non-TDLN immune contexture revealed prominent shifts in immune cell frequencies and phenotypes, including a decrease in T-cell frequencies and a marked increase in CD169+ LN-macrophages in the TDLN. Systemic macrophage targeting using CSF1R-kinase inhibition or CEL effectively minimized LN-macrophage subsets as well as TAMs, therefore preventing the specific interrogation of LN-macrophage biology during tumor growth and in the context of immune activation. Conversely, titrating CEL doses down to 5% of the total dosing volume injected i.pl. allowed for the specific depletion of LN-macrophages while leaving systemic macrophages undisturbed. LN-macrophages limited the presence of migratory ex vivo activated and tumor-loaded DCs, as indicated by increased counts of CFSE+ DCs in the in the TDLN following adoptive transfer in CEL-pretreated mice. Depletion of TDLN-macrophages increased LN- and blood frequencies and activation status of DC-induced CD8+ T-cells and CD4+ T-helper cells, indicative of enhanced antitumor immunity. Furthermore, whereas DC-induced immune activation alone was insufficient to impede tumor growth and prolong survival, elimination of LN-macrophages prior to DC-administration enabled the successful impediment of tumor growth. Interestingly, interrogation of tumor-infiltrating T-cell (TIL) phenotype and distribution showed a robust increase in CD4+ T-helper TILs and a concurrent decrease in CD8+ TIL- exhaustion phenotype, as evidenced by diminished PD-1 and LAG-3 expression. Altogether, these results indicate an important immune inhibitory role for LN-macrophages in settings of immune activation in the solid tumor setting. These findings further increase our knowledge of where and how macrophages exert their immune-inhibitory functions and allow for better insight into the underpinnings of immunotherapy resistance.
Citation Format: Floris F. Dammeijer, Mandy van Gulijk, Melanie M. Lukkes, Menno van Nimwegen, Rudi W. Hendriks, Thorbald T. van Hall, Heleen H. Vroman, Joachim J.G.J.V. Aerts. Depletion of macrophages in the tumor-draining lymph node enhances dendritic cell-induced antitumor immunity and survival [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A165.